1. Field of the Invention
This invention relates to devices and techniques for chemical processing multiple mixtures, and more particularly, to an apparatus and method for sealing and purging multi-well reactors useful in high throughput protein analysis.
2. Discussion
Pharmaceutical and biomedical researchers continually seek new methods for rapidly identifying therapeutically important proteins. This interest has fueled an ongoing development of high throughput methods and instruments for carrying out protein analysis--an important element of an emerging scientific discipline known as proteomics. The field of proteomics generally involves systematic isolation, identification, and characterization of proteins present in biological samples. Proteomics typically employs two-dimensional gel electrophoresis (2DE) to separate complex mixtures of proteins. Once separated, individual proteins are subsequently identified and characterized based on their role in disease processes or performance in drug assays.
Steps in a typical proteomic protocol include: (1) solubilizing proteins using detergents to release proteins trapped in cells or tissue; (2) separating the proteins using two dimensional gel electrophoresis; (3) staining the gel to locate individual proteins; and (4) scanning the stained gel for proteins of interest. Scanning may include, for example, selecting proteins that occur in diseased tissue but are absent in healthy tissue. The protocol also includes: (5) picking or removing portions of the gel containing the proteins of interest; (6) breaking down the proteins removed from the gel into protein fragments (polypeptide residues); and (7) measuring the size (molecular weight) of the isolated proteins and residues using mass spectroscopy. Since proteins are heat labile, the mass spectroscopy technique usually employs a soft ionization technique, such as fast atom bombardment (FAB), field desorption (FD), atmospheric pressure ionization (API), or matrix-assisted laser desorption (MALDI). The last step in the protocol is (8) identifying protein fragments by comparing their sizes with other peptide (amino acid) sequences found in public and private databases. Once identified, researchers can evaluate the role of each protein in a disease process, and target the protein for drug intervention.
Currently, the steps in a proteomic analysis are done in a sequential and modular fashion. The output from one step is transferred manually to the next step, which makes the technique slow and cumbersome. It appears that recent advances in robotics, software design, and computer technology, could improve the sample throughput, rate of analysis, and reliability of the analysis. However, other problems remain.
The digestion step (6) is typically carried out in multi-well reactors, such as 96-well and 384-well microtiter plates. Microtiter plates comprise an array of depressions formed on a generally planar surface of a tray, and can be adapted to allow thermal processing of samples. Liquid samples, reagents, buffers, and the like, are normally added or removed from the wells by pipette, which may be automated using laboratory robotic systems. Solids may be placed in the wells, or may result from chemical reaction or changing conditions within a liquid sample (e.g., precipitation). In solid-liquid mixtures, one difficulty arises when using a pipette to purge the liquid-phase while retaining the solid-phase within the wells, as would occur, for example, when washing a solid sample with a liquid or when removing liquid-phase reactants and side products following chemical reaction. Although easy to add, liquids are hard to remove thoroughly from the wells because vacuum generated by the pipette is insufficient to overcome capillary forces that confine the liquid within the interstices of the solid or against the walls of the wells. The ability to thoroughly purge liquid from the wells is an important and common requirement of many processes, including protein digestions.
One way to ensure thorough removal of the liquid phase is to seal the wells and to apply sufficient pressure within the wells to purge liquid through holes provided in the bottom of each of the wells. The size of the holes is small enough to prevent passage of the solid phase during liquid purging; in the absence of an applied pressure, capillary forces are sufficient to retain the liquid phase in the wells. In this system, the desired product may be either the solid phase, which is retained in the wells, or the liquid phase, which is purged from the wells and can be collected in a second microtiter plate for example.
A robotic liquid handling system can be used to transfer reagents to the wells using a syringe pump coupled to a probe. The probe is comprised of inner and outer, coaxial cylindrical tubes. The inner tube, which extends outward from the end of the outer tube, aspirates or dispenses liquid; the outer tube dispenses gas. Before processing, each of the wells is sealed with a plastic cap having a tapered hole, which is sized to allow the probe to access the interior of the well. During the addition of liquid, the probe is inserted partway into the tapered hole so that air displaced by the liquid may escape from the well through the gap between the wall of the hole and the portion of the inner tube that extends beyond the end of the outer tube. When pressurizing the well, the probe is fully inserted in the tapered hole so that a substantially gas-tight seal is formed between the wall of the tapered hole and the exterior surface of the outer tube.
Although caps can work well, they have shortcomings. For example, large numbers of individual caps are difficult to handle and hard to seat properly in the wells. Although the caps can be manufactured by injection molding, the caps are relatively expensive unless groups of caps are molded in a single shot. But even when injecting molding large numbers of caps in a single shot, the tooling costs for multi-cavity molds can be high. Also, because the outer tube has to seal against the wall of the tapered hole, the caps wear out and must be replaced.
The present invention overcomes, or at least reduces, one or more of the problems set forth above.